U.S. patent number 6,156,882 [Application Number 09/105,596] was granted by the patent office on 2000-12-05 for antibody 4g8b4b12.
This patent grant is currently assigned to Eberhard-Karls-Universitat Tubingen. Invention is credited to Hans-Jorg Buhring, Irene Rappold.
United States Patent |
6,156,882 |
Buhring , et al. |
December 5, 2000 |
Antibody 4G8B4B12
Abstract
The invention relates to a monoclonal antibody specifically
binding to the human FLT3/FLK2 receptor protein. The invention
further relates to hybridoma cells producing such an antibody, as
well as to a method for generation of such hybridoma cells. The
monoclonal antibody is the antibody produced and released by
hybridoma cells deposited under No. DSM 2249 at the German
Collection of Microorganisms and Cell Cultures (DSMZ) and
designated as 4G8B4B12.
Inventors: |
Buhring; Hans-Jorg (Tubingen,
DE), Rappold; Irene (Tubingen, DE) |
Assignee: |
Eberhard-Karls-Universitat
Tubingen (Tubingen, DE)
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Family
ID: |
7834132 |
Appl.
No.: |
09/105,596 |
Filed: |
June 26, 1998 |
Foreign Application Priority Data
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Jun 30, 1997 [DE] |
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197 27 814 |
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Current U.S.
Class: |
530/388.1;
435/326; 530/387.1; 530/388.2; 530/388.22; 530/391.1;
530/391.3 |
Current CPC
Class: |
C07K
14/70596 (20130101); C07K 16/2896 (20130101); G01N
33/56972 (20130101); A61K 38/00 (20130101); G01N
2333/70582 (20130101); G01N 2333/71 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/705 (20060101); C07K
16/28 (20060101); C07K 16/18 (20060101); G01N
33/569 (20060101); A61K 38/00 (20060101); C12N
009/64 (); C07K 016/00 () |
Field of
Search: |
;435/326,252.3
;530/387.1,388.1,388.2,388.22,388.7,391.1,391.3 ;424/130 |
Foreign Patent Documents
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196 08 769 C1 |
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Apr 1997 |
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DE |
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WO 95/07438 |
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Mar 1995 |
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WO |
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Other References
Rappold I. et al., "Epitope Recognition, Functional Aspects, and
Reactivity of Anti-FLT3-FLK2 Antibodies", Tissue Antigens, vol. 48
No. 4-2 (1996) p. 399 (XP002080668). .
Rosnet O. et al., "Human FLT3/FLK2 Receptor Tyrosine Kinase is
Expressed at the Surface of Normal and Malignant Hemotopoietic
Cells", Leukemia, vol. 10 No. 2, (1996) pp. 238-248 (XP002080669).
.
Goldstein, N. "71E1, Anti-FLK-2 Tyrosine Kinase Receptor",
Hybridoma, vol. 14 No. 5, (1995) p. 513 (XP002080670). .
Rose, C. et al., "Isolation and Characterization of a Monoclonal
Antibody Binding to the Extracellular Domain of the FLK-2 Tyrosine
Kinase Receptor", Hybridoma, vol. 14 No. 5, (1995) pp. 453-459
(XP002080671). .
Rappold I. et al., "Functional and Phenotypic Characterization of
Cord Blood and Bone Marrow Subsets Expressing FLT3 (CD135) Receptor
Tyrosine Kinase", Blood, vol. 90 No. 1, (1997) pp. 111-125
(XP002080672). .
Kimmel et al (J. Neurosurg, 66:161-171), 1987. .
Hird et al (in "Genes and Cancer", Carney et al., Eds., John Wiley
and Sons Ltd, 83-89), 1990. .
Freshney (Culture of Animal Cells, A Manual of Basic Tecnique, Alan
R. Liss, Inc., New York, p. 4), 1983. .
Dermer (Bio/Tchnology, 12:320), 1994..
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Primary Examiner: Ungar; Susan
Attorney, Agent or Firm: Oppenheimer, Wolff & Donnelly,
LLP Hamrick; Claude A. S.
Claims
What is claimed is:
1. A monoclonal antibody binding specifically to the human
FLT3/FLK2 receptor protein and being produced and released by
hybridoma cells that are deposited at the International Depositary
Authority DSMZ-Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH Mascheroder Weg 1b, D-38124 Braunschweig,
Germany, as of Dec. 19, 1995 under No. DSM ACC2249 in accordance
with the Budapest Treaty, and designated 4G8B4B 12.
2. Hybridoma cells deposited at the International Depositary
Authority DSMZ-Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH Mascheroder Weg 1b, D-38124 Braunschweig,
Germany, as of Dec. 19 1995 under No. DSM ACC2249 in accordance
with the Budapest Treaty, and designated 4G8B4B 12.
3. A composition comprising an antibody according to claim 1 and a
pharmaceutically acceptable carrier.
4. The composition according to claim 3, wherein the antibody is
coupled to a labeling marker.
5. Kit comprising an antibody according to claim 1.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a monoclonal antibody directed against the
human tyrosine kinase receptor FLT3/FLK2.
The tyrosine kinase receptor FLT3/FLK2 plays an important role in
early hematopoiesis. It naturally occurs in blood stem cells and
early lymphoid and myeloid blood progenitor cells and interacts
with a growth factor FLT3-ligand (FL), that particularly stimulates
the recruitment and proliferation of these cells (Matthews et al.,
Cell 65:1143, 1991; Small et al., Proc. Natl. Acad. Sci. USA
91:459, 1994; Lyman et al., Blood 83:2795, 1994; Muench et al.,
Blood 85:963, 1995; Hannum et al., Nature 368:643, 1994; Hirayama
et al., Blood 85:1762, 1995).
2. Description of the Related Art
The indirect detection mediated by an antibody specifically binding
to the receptor represents a safe and quick method to qualitatively
and quantitatively detect membrane bound receptors. The specific
antibody can be labeled either directly or indirectly, using this
label for identification and quantitative determination of the
antibody or the bound receptor, respectively. Particularly,
fluorescent dyes or radioactive agents can be used as labels.
Such an antibody can also be coupled to special therapeutically
active agents and therefore renders possible a targeted cellular
treatment, which is particularly required for the treatment of
tumorous diseases.
Antibodies directed against FLT3/FLK2 receptor protein have already
been described, however, these antibodies are directed against the
murine FLT3/FLK2 receptor protein. Therefore, lacking the required
specificity, these antibodies are not suitable for the treatment of
human cells.
A monoclonal antibody binding specifically to the native unmodified
human FLT3/FLK2 receptor protein, is produced and released by
hybridoma cells that were deposited on Dec. 6, 1997 at the
International Depository Authority DSMZ-Deutsche Sammlung Von
Mikroorganismen Und Zellkulturen Gmbh (German Collection of
Microorganisms and Cell Cultures, Ltd. (DSMZ)), Mascheroder Weg 1b,
D-38124 Braunschweig Germany under No. DSM ACC2248 at the German
Collection of Microorganisms and Cell Cultures Ltd. (DSMZ) in
accordance with the provision of the Budapest Treaty. It has been
given the designation BV10A4H2. This antibody is subject matter of
German Patent DE 196 08 769 titled "antibody BV10A4H2".
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a
further antibody specifically binding to the native unmodified
human FLT3/FLK2 receptor protein and which is available in
practically unlimited amounts.
This object is achieved by providing a monoclonal antibody binding
specifically to the native unmodified human FLT3/FLK2 receptor
protein and being produced and released by hybridoma cells that
were deposited under No. DSM ACC2249 at the German Collection of
Microorganisms and Cell Cultures Ltd. (DSMZ) in accordance with the
provisions of the Budapest Treaty. The antibody has been given the
designation 4G8B4B12.
With the antibody according to the invention, a monoclonal antibody
has been provided for the first time that is reproduceable in a
standardized manner and can thus potentially be produced in
unlimited amounts and that binds specifically to a respective
special extracellular epitope of the human receptor protein
FLT3/FLK2.
The antibody according to the invention permits a targeted
detection and modulation of cells comprising an extracellular
domain of the human FLT3/FLK2 receptor protein. It therefore
provides to physicians and research personnel a sofar unique and
variably applicable means for on the one hand detecting such cells,
both in cell culture and in the patient's organism, and on the
other hand manipulating such cells, if desired, either by means of
the antibody as such, or by specific reagents coupled to it.
The invention further relates to hybridoma cells producing a
monoclonal antibody directed against the FLT3/FLK2 receptor protein
and being deposited under the number DSM ACC2249 at the German
Collection of Microorganisms and Cell Cultures Ltd. (DSMZ)
according to the Budapest Treaty and producing the antibody
designated 4G8B4B12.
The invention further relates to a method for producing hybridoma
cells synthesizing and releasing an antibody directed against the
native unmodified FLT3/FLK2 receptor protein. This method comprises
the steps generally known in the art, as described for example by
Buhring et al. in Hybridoma 1991, Vol. 10, No. 1, pp. 77-78:
1. Immunizing or sensitization of an animal, preferably a mouse of
the Balb/c line, with the antigen or immunogen;
2. collecting the antibody-producing cells, preferably the
lymphocytes of the spleen of that animal;
3. fusion of these antibody-producing cells with a stable,
immortalized cell line, preferably a myeloma cell line, to
hybridoma cells; and
4. isolation and multiplication (cloning) of such hybridoma cells
that secrete an antibody binding to the antigen.
The method according to the invention is characterized by the fact
that the animal is immunized with cells of the murine cell line
BA/F3 that had previously been transfected with the complete
cDNA-sequence of the human FLT3/FLK2 receptor (BA/F3-huFLT3).
It is advantageous that the cells of this cell line BA/F3-huFLT3
present a strong expression of FLT3/FLK2 receptor protein, as has
been shown during the experiments leading to these transfected
BA/F3-huFLT3 cells.
When screening hybridoma cells producing antibodies specific for
FLT3/FLK2 receptor protein, it is preferred, if only those isolated
and cloned hybridoma cells are selected which produce antibodies
having a specificity for the transfected BA/F3-huFLT3 and showing a
negative reaction with cells of the non-transfected BA/F3 cell
line.
The invention also relates to the use of the inventive monoclonal
antibody directed against the FLT3/FLK2 receptor protein for
diagnostic and/or therapeutic treatment of tumors, particularly of
malignant hematopoietic cells as for example lymphoid and myeloid
leukemia cells.
Surprisingly, it has been found that most malignant hematopoietic
cells as, for example, lymphoid and myeloid leukemia cells comprise
a comparably high content of FLT3/FLK2 receptor protein. An
antibody according to the invention coupled to a means for
detection, for instance a radioactive marker, indirectly binds this
detection means to the respective cells and thus allows the direct
detection of the cells, for example using x-ray
diagnostic/scintigraphic methods. Therefore, a very early diagnosis
of tumors is possible, even in vivo.
In an analogous way the antibody may be coupled to a
therapeutically active agent and therefore allows a direct and
targeted modulation or even elimination of FLT3/FLK2 receptor
protein carrying cells, particularly leukemia cells.
In order to facilitate the therapeutic and/or diagnostic
application of the antibody according to the invention, the
antibody may be mixed in a pharmaceutical composition with adequate
accessory substances. Consequently, the invention also relates to a
pharmaceutical agent for diagnostic and/or therapeutic treatment of
tumors, comprising an antibody produced and released by the
hybridoma cells deposited under No. DSM ACC2249 at the German
Collection of Microorganisms and Cell Cultures Ltd. (DSMZ).
Using an antibody according to the invention, cells carrying the
human FLT3/FLK2 receptor protein may be detected in a suspension of
different (human) cells using the methods known in the art, for
instance the enzyme-linked immunosorbent assay, ELISA, or the radio
immuno assay, RIA. The present invention therefore also relates to
a kit for the detection of human FLT3/FLK2 receptor protein,
comprising a monoclonal antibody produced by the hybridoma cells
deposited under No. DSM ACC2249 at the German Collection of
Microorganisms and Cell Cultures Ltd. (DSMZ).
In connection with the present invention, it has surprisingly been
found that the monoclonal antibody designated 4G8B4B12, produced by
the hybridoma cells deposited under No. DSM ACC2249 at the German
Collection of Microorganisms and Cell Cultures Ltd. (DSMZ) binds to
human stem cells.
The invention therefore also relates to the use of the antibody
4G8B4B12 for detection of hematopoietic cells, as well as to a kit
for the detection of hematopoietic cells, comprising the antibody
4G8B4B12. It is thereby possible to separate undifferentiated
CD34.sup.+ subpopulations and to select and purify cells from bone
marrow for functional analyses.
Further advantages can be taken from the following description.
It is understood that the afore-mentioned features and those to be
explained below can be used not only in the specific combinations,
but also in other combinations or alone without going beyond the
scope of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be described hereafter with reference to certain
applications and embodiments, in combination with the drawings, in
which:
FIG. 1 shows a histogram A of flow cytometer measurements of 4G1
cells, incubated with ligand and without antibody, and a histogram
B of a flow cytometer measurement of 4G1 cells, incubated with
ligand and antibody 4G8B4B12 (therein designated 4G8); and
FIG. 2 shows three histograms of flow cytometer measurements on
BV-173 cells, preincubated at B with a control antibody and then
incubated with the antibody 4G8B4B12 (therein 4G8) and the ligand,
at C directly with the antibody 4G8B4B12 (therein 4G8) according to
the invention and with the ligand, and at A with the ligand
alone.
DETAILED DESCRIPTION
EXAMPLE 1
Generation and characterization of monoclonal antibodies directed
against the FLT3/FLK2 receptor protein
Cells of a murine cell line BA/F3 that had previously been
transfected with the complete cDNA for the human FLT3/FLK2 receptor
protein (BA/F3-huFLT3) are used as antigen.
To generate these cells the complete coding sequence of the human
cDNA for FLT3/FLK2 receptor protein was cloned into the mammalian
expression vector pCD-SR and introduced together with a plasmid
conferring G418 resistance into the interleukin-3 dependent BA/F3
cells.
Transfected cells expressing the FLT3/FLK2 receptor were isolated
and concentrated on the basis of their G418 resistance and
according to their growth in the presence of recombinant FLT3/FLK2
receptor protein-ligand (FL) (Lyman et al., Cell 75:1157, 1993).
One clone, 4G1, was selected after limited dilution and tested for
its ability to express FLT3/FLK2 receptor protein. For this purpose
cells of clone 4G1 were first labeled with radioactive methionine
(.sup.35 S) and then incubated with a rabbit antiserum directed
against the inserted domain of the tyrosine kinase of the receptor
protein FLT3/FLK2. The thus generated immunoprecipitate was
analyzed using polyacrylamide gel electrophoresis.
The thus confirmed BA/F3-HuFLT3 cells of the clone 4G1 were used as
the antigen.
Eight weeks old Balb/c mice are immunized intraperitoneally twice,
at intervals of 10 days, with 10.sup.7 cells of the clone 4G1. Four
days before the fusion, 5.times.10.sup.5 cells are administered
directly into the spleen in order to reinforce the immune
response.
The formation of antibodies in the organism of the mouse is tested
by screening the blood serum of the respective animal for binding
properties with the antigen using the ELISA test well-known to any
person skilled in the art.
Approximately 3 weeks later the lymphocytes of the successfully
immunized animal are collected by removing the animal's spleen and
disintegrating it into a cell suspension.
The suspended spleen cells are fused with myeloma cells of the
known cell line SP2/0 in the presence of polyethylene glycol. The
fusion culture is cultivated in a medium containing hypoxanthine,
aminopterine and thymidine (HAT), herein HAT-RPMI-1640, in which
only hybrid cells grow as these have both the property of myeloma
cells to divide infinitely, and the property of the
antibody-producing lymphocytes to grow in a medium containing
HAT.
Following fusion, the cells are plated into microtiter plates and
are incubated at 37 C in the presence of 5% CO.sub.2.
The culture supernatants are screened in a flow cytometer after
10-14 days on cells of the clone 4G1. In a second step the
supernatants were tested for reactivity with the cells of the cell
line BA/F3, as these cells do not express antigens. Hybridoma cells
producing antibodies specific for 4G1 cells are selected, isolated
and cultivated, i.e. cloned, according to the known limited
dilution method.
Positively reacting hybridoma cell cultures are subjected to
further cultivation, the antibodies are concentrated, purified and
characterized.
The monoclonal antibody 4G8B4B12 was obtained using the
above-mentioned screening strategy. Using a phycoerythrine
(PE)-conjugated anti-isotype-specific antiserum by direct
immunofluorescence using flow cytometry, the isotype IgG1 was
determined.
Production, purification and characterization of the antibodies
were carried out using methods generally known in the art.
The antibody 4G8B4B12 produced by the hybridoma cells deposited
under No. DSM ACC2249 at the German Collection of Microorganisms
and Cell Cultures Ltd. (DSM), exhibits the following characteristic
features:
______________________________________ Immunoglobulin class: IgG1
specific binding affinity to: human FLT3/FLK2 receptor protein
(extracellular domain) ______________________________________
EXAMPLE 2
Identification of the antigen recognized by the monoclonal antibody
4G8B4B12.
Identification of the antigen was carried out by testing binding to
cells with and without FLT3/FLK2 receptor.
A sample (A) containing mouse fibroblasts of the cell line
BA/F3-huFLT3 generated according to example 1 and transfected with
the human cDNA for FLT3/FLK2 receptor, was incubated with the
antibody 4G8B4B12 of the invention.
As a control, a sample (B) containing mouse fibroblasts of
non-transfected cell line BA/F3 was equally incubated with the
antibody 4G8B4B12 in the same concentration as sample (A).
Both samples were labeled with an anti-IgG1-PE antiserum and then
analyzed in a flow cytometer.
Binding of the antibody 4G8B4B12 could be shown exclusively for
sample (A). This means that the antibody 4G8B4B12 according to the
invention had specifically bound to those cells transformed with
the cDNA for human FLT3/FLK2 receptor and therefore expressing the
human FLT3/FLK2 receptor.
EXAMPLE 3
Identification of the monoclonal antibody 4G8B4B12 as a
non-antagonistic and slightly agonistic reacting antibody
In tyrosine kinase receptors such as the FLT3/FLK2 receptor
receptor activation initiated by ligand binding first leads to
formation of receptor dimer complexes and their internalization
into the cell. Some antibodies can simulate the ligand, i.e. they
act agonistically, and therefore lead to such a receptor dimer
formation and internalization as well (Buhring et al., Cancer Res.
53: 4424, 1993).
A sample (A) containing 4G1 cells (obtained according to example 1)
was incubated with 200 ng/ml biotinylated FLT3 ligand-biotine and
streptavidin-phycoerythrine (SA-PE).
A second sample containing 4G1 cells (B) was initially incubated
with 7 .mu.g/ml of the antibody 4G8B4B12 for 30 minutes and
subsequently treated as sample (1A).
Both samples were analyzed in the flow cytometer. The resulting
histograms are given in FIG. 1.
Both histograms show equally strong signals. This means that the
antibody 4G8B4B12 according to the invention has no inhibiting
effect to the ligand binding and therefore does not act
antagonistically (Buhring et al., Cancer Res. 53: 4424, 1993).
For detection of the agonistic reaction a sample (B) containing
cells of the hematopoietic pro-B cell line BV-173 (DSM ACC 20;
freely accessible) was preincubated with a control IgG1 antibody
for 2 hours at 37 C, then incubated with the antibody 4G8B4B12 of
the invention and finally incubated with anti-IgG1-PE
antiserum.
Another sample (C) was initially incubated with the antibody
4G8B4B12 and subsequently treated as sample (B).
A control sample (A) was initially incubated with 100 ng/ml
FLT3/FLK2 receptor ligand and then treated as sample (B).
All three samples were analyzed in the flow cytometer. The
resulting histograms are given in FIG. 2. Compared to histogram B
histogram C shows a signal reduced by approximately 33%. This means
that the antibody 4G8B4B12 according to the invention has caused
internalization of some FLT3/FLK2 receptors and therefore acts
slightly in an agonistic way (ligand stimulating).
Compared to histogram B histogram A shows a signal reduced by 90%
caused by internalization of the FLT3/FLK2 receptors by naturally
occurring ligands.
EXAMPLE 4
Use of the monoclonal antibody 4G8B4B12 for detection of
subpopulations of blood stem cells
A sample containing freshly obtained bone marrow cells was
separated in a Ficoll-Hypaque density gradient and the normal,
mononuclear bone marrow cells were isolated and concentrated.
These mononuclear bone marrow cells were then incubated with a
PE-conjugate of the antibody 4G8B4B12 of the invention and with
known fluorescein-isothiocyanate (FITC)-conjugated antibodies
directed against the stem cell antigens HLA-DR, CD34 and CD62L,
respectively, CD33 occurring in myeloid cells, CD71 occurring in
erythroid and progenitor cells, CD10 and CD19 occurring in B-cells,
respectively and CD3 and CD7, occurring in T-cells,
respectively.
The cells were analyzed in a flow cytometer.
The results of the analysis show:
a strong co-expression of FLT3/FLK2 with the stem cell markers
CD34, HLA-DR and CD62L (55% and 100% and 60%, respectively),
a weaker co-expression of the myeloid cell marker CD33 and the
erythroid and progenitor cell markers CD10 and CD19 (60% and 20%
and 20%, respectively), and
no co-expression with the T-cell markers CD7 and CD3.
This means that blood stem cells as well as myeloid cells and
progenitor cells of the B-line can be identified and particularly
differentiated from T-cells using the antibody 4G8B4B12 of the
invention.
A 3-color analysis was carried out for characterization of
subpopulations of the CD34.sup.+ stem cell population as
follows:
The mononuclear cells were incubated with a PE-conjugate of the
antibody 4G8B4B12 of the invention, with a known FITC-conjugated
antibody directed against CD34, and a known biotine-conjugated
antibody directed against CD117, and subsequently analyzed in the
flow cytometer.
The obtained results show that 23% of the CD34.sup.+ cells also
express the receptor protein FLT3/FLK2 and 50% of the cells of this
subpopulation additionally comprise the protein CD117.
Analyses on CD34.sup.++ and CD34.sup.+++ cells show that the number
of cells of the subpopulation of FLT3/FLK2.sup.+ /CD117.sup.+ cells
decreases when CD34 expression is decreasing (from CD34.sup.+++ via
CD34.sup.++ up to CD34.sup.+), i.e. with ongoing development by the
blood system cells, while the subpopulation of FLT3/FLK2.sup.+
/CD117.sup.- cells is increasing.
It is therefore possible to isolate two new subpopulations of the
CD 34-positive population of blood stem cells using the antibody
4G8B4B12 of the invention, namely the subpopulation FLT3/FLK2.sup.+
/CD117.sup.+ and the subpopulation FLT3/FLK2.sup.+
/CD117.sup.-.
EXAMPLE 5
Use of the monoclonal antibody 4G8B4B12 for detection of leukemic
blasts of the myeloid and B-lymphocytic line
By centrifugation in a Ficoll gradient, a pure (>90%) population
of leukemic blasts was obtained from bone marrow or peripheral
blood of patients suffering from leukemia.
The cells were classified according to the French-American-British
(FAB) classification (Bennet et al., Ann. Intern. Med. 103: 620,
1985) and this classification was confirmed by phenotypic
analyses.
The cells were incubated with the antibody 4G8B4B12, the bound
antibody was labeled with the PE-conjugated anti-IgG1 antiserum and
the cells were analyzed in the flow cytometer.
In most of the blast-populations of patients suffering from acute
myeloid and/or B-lymphocytic leukemia a positive reaction and
therefore the presence of FLT3/FLK2 receptor protein was
detected.
Further to these analyses using freshly isolated leukemia cells,
samples of megakaryoblastic cell lines, myeloid/monocytic cell
lines, and B-cell lines were tested.
The cells of all samples were equally incubated with the antibody
4G8B4B12, the bound antibody was labeled with a PE-conjugated
anti-IgG1 antiserum and the cells were analyzed in the flow
cytometer.
A positive reaction and therefore an expression of FLT3/FLK2
receptor protein was found on most of the cell lines of the
B-lymphocytic line, and on few of the myeloid line.
The antibody 4G8B4B12 of the invention has therefore proven to be a
particularly well-suited means for the detection and identification
of malignant hematopoietic cells of the myeloid and B-lymphocytic
line.
* * * * *